Neural circuits of complex brains are frequently organized into parallel layers (laminae), with distinct populations of afferents innervating specific layers. Such layer specificity is widely observed in vertebrate and invertebrate brains, and it is likely a major determinant of synaptic specificity in the central nervous system. Our research group studies the development of layer-specific connections using Drosophila visual system as a model. The Drosophila compound eye consists of approximately 800 ommatidia, each containing three types of photoreceptor neurons (R1-6, R7, and R8). The R1-6 neurons respond to green light and connect to the first optic ganglion (the lamina), whereas the R7 and R8 are sensitive to ultraviolet and blue lights, respectively, and connect to the second optic ganglion (the medulla). The medulla is subdivided into ten layers (M1-10). The information from these three types of photoreceptor neurons is directed to distinct medullar layers: the R7 and R8 directly connect to the M6 and M3 layers, respectively, while the laminal neurons (L1-5) relay R1-6 input to multiple medullar layers. The establishment of layer-specific connections by different afferents in the medulla is critical for various visual functions, including color vision. We have been focusing on the development of R7-M6 connections. Using single-cell mosaic techniques, we first determined the developmental processes of R7 target selection. We found that R7 axons select their target layer in two distinct stages. During the late larval and early pupal stage (the first target-selection stage), R7 neurons sequentially differentiate and project axons into the R7-temporary layer where they remain for 1-2 days. During the late pupal stage (the second target-selection stage), all R7 growth cones regain motility and synchronously project into the destined layer, the R7-recipient layer. The characterization of the development of R7-brain connections provides a framework to study the isolated mutants. In a genetic screen based on innate color-discrimination behavior, we identified 5 loci that affect R7 connectivity. These are three molecularly characterized loci, N-cadherin, receptor phosphatase LAR, and milton, as well as two novel loci, pex (premature extension) and ovs (overshoot). These mutants provide molecular handles for dissecting the mechanisms of R7 layer-selection. Removing N-cadherin in R7 axons results in their failure to extend into the R7-temporary layer at the early pupal stage, and subsequently at the adult stage, mis-connect to the R8-recipient layer. The Drosophila N-cadherin belongs to a unique type of evolutionary conserved classical cadherins that have large complex extracellular domains and catenin-binding cytoplasmic domains. To determine the functional domains of N-cadherin, we conducted a structure-function analysis. We found that the cytoplasmic domain of N-cadherin is not essential for mediating homophilic interaction in cultured cells, and is largely dispensible for layer-specific targeting of R7 axons in vivo. However, the N-cadherin cytoplasmic domain, and hence its catenin-binding activity, is required for maintaining proper morphology of R7 growth cones. Domain swapping with the extracellular domain of N-cadherin2, a related but non-adhesive cadherin, revealed that the N-cadherin extracelluar domain is required for both adhesive activity and R7 targeting. Together, these results suggest that N-cadherin mediates adhesive interactions and not cytoplasmic signaling to regulate R7 target selection. The mutations, pex and ovs, affect R7 target selection in the opposite way to what N-cadherin and LAR mutations do. In contrast to N-cadherin and LAR mutant R7 axons, which retract to the superficial R8-recipient layer, pex and ovs mutant R7 axons overshoot the target region. Interestingly, ovs mutant R7 axons overshoot the R7-recipient layer and terminate at a deeper layer, while pex mutant R7 axons extend into the neighboring target area occupied by other R7 termini or loop back to more superficial layers. Developmental analysis revealed complex pex phenotypes: pex mutant R7 axons overshoot the R7-temporary layer at the early pupal stage but subsequently retract to the R7-temporary at the mid-pupal stage. However, during the late pupal stage, pex mutant R7 growth cones regain motility and extend into the neighboring target region. We hypothesize that pex and ovs provides the opposing (or balancing) effect on the R7 growth cones, presumably by counteracting N-cadherin or LAR function. We are currently cloning pex and ovs in the hope that their molecular identify will provide insight into the regulatory mechanisms of R7 target selection.